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DNA fluorescence analysis method based on the interaction between hypericin and 1-pyrene butyric acid

A fluorescence analysis and magnetic separation technology, applied in the field of DNA fluorescence analysis, can solve the problems of difficult single nucleotide polymorphism detection, high background noise of electrochemical method, low hybridization efficiency, etc., achieve good linear response, simple method, The effect of short analysis times

Inactive Publication Date: 2019-03-05
NANJING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, the method of using the electroactivity of ferrocene to detect DNA has been widely used. However, the background noise of the electrochemical method is large, the reproducibility is poor, and it is difficult to be used for single nucleotide polymorphism detection. The hybridization efficiency of the DNA hybridization probe used in the method is lower than that of peptide nucleic acid, morpholine oligonucleotide and other probes, and it is not suitable for DNA detection of base mismatches

Method used

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  • DNA fluorescence analysis method based on the interaction between hypericin and 1-pyrene butyric acid
  • DNA fluorescence analysis method based on the interaction between hypericin and 1-pyrene butyric acid
  • DNA fluorescence analysis method based on the interaction between hypericin and 1-pyrene butyric acid

Examples

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Effect test

Embodiment 1

[0029] Example 1: Ultraviolet and fluorescence spectrum characterization of fluorescent probes introduced on the surface of SA-MB through the coordination of zirconium ions and the π-π stacking between Hyp and PBA.

[0030] Using the DNA fluorescence analysis based on the π-π stacking effect between Hyp and PBA described in the present invention, suspend 0.5 mg / mL SA-MB in PBS buffer, add 1 μM MO modified with biotin at the 3' end, The sequence is 5'-AAC CAT ACA ACCTAC TAC CTC A-Biotin-3'. After reacting for 60 minutes, perform magnetic separation and wash with PBS buffer several times, then add 100nM tDNA (dissolved in TE buffer), the sequence is 5'- TGA GGT AGT AGG TTG TAT GGT T-3', after hybridization for 60min, perform magnetic separation and wash with Tris-HCl buffer several times, then add 4mM ZrOCl 2 ·8H 2 O solution (dissolved in 60% ethanol), after coordination reaction for 30min, carry out magnetic separation and wash with 60% ethanol several times, then add 2mM PBA...

Embodiment 2

[0031] Example 2: Time optimization of π-π stacking interaction between Hyp and PBA.

[0032] Using the DNA fluorescence analysis based on the π-π stacking interaction between Hyp and PBA according to the present invention, the operation steps are the same as the above-mentioned Example 1. Among them, 100nM tDNA fragment with the sequence of 5'-TGA GGT AGT AGG TTG TAT GGT T-3' was selected as the target analyte, and the π-π stacking time was changed to 10, 20, 30, 40, 50, 60, 70 , 80min, analyze the influence of reaction time on the DNA fluorescence analysis result of the present invention, its analysis result is as attached image 3As shown, where a is the change of the fluorescence spectrum, b is the change of the peak value, it can be seen from the figure that when the π-π stacking time is 10, 20, 30, 40, 50 min, the fluorescence intensity increases significantly with the increase of time , and after 50 min of reaction, the fluorescence intensity remained stable with the i...

Embodiment 3

[0033] Example 3: Effect of water content on π-π stacking.

[0034] Using the DNA fluorescence analysis based on the π-π stacking interaction between Hyp and PBA according to the present invention, the operation steps are the same as the above-mentioned Example 1. Among them, 100nM of the tDNA fragment with the sequence of 5'-TGA GGT AGT AGG TTG TAT GGT T-3' was selected as the target analyte, and the water content in the π-π stacking reaction was changed to 0%, 10%, 20%, 30% %, 40%, 50%, 60%, 70%, 80%, 90%, 100%, to analyze the effect of water content on π-π stacking, the analysis results are as attached Figure 4 As shown, where a is the comparison of fluorescence spectra, and b is the comparison of peak values. It can be seen from the figure that with the increase of water content, the fluorescence intensity decreases significantly. After the water content exceeds 80%, the fluorescence intensity almost drops to zero, so in In the actual detection process, it is necessary t...

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Abstract

The invention discloses a DNA fluorescence analysis method based on pi-pi stacking interaction between hypericin and 1-pyrenebutyric acid. The method comprises the following steps: hybridizing a capture probe morpholino oligonucleotides covalently linked to the surface of a streptavidin magnesphere with a target DNA fragment, connecting 1-pyrenebutyric acid to a DNA skeleton by using the coordination effect of zirconium ions, and introducing hypericin with strong fluorescence by using the pi-pi stacking interaction between hypericin and 1-pyrenebutyric acid. A fluorescence probe is introduced through the coordination effect of transition metal ions and the pi-pi stacking interaction between aromatic rings, so the method is simple, the binding effect of the fluorescence probe is good, and the measured fluorescence intensity is related with the target DNA; and the fluorescence analysis method realizes highly-sensitive and highly-selective detection of the DNA fragment, and can be applied to detection of a DNA sample in the fields of molecular identification, clinic diagnosis, environment monitoring and food safety.

Description

technical field [0001] The invention belongs to the application field of fluorescence analysis, in particular to a DNA fluorescence analysis method based on π-π stacking between Hyp and PBA. Background technique [0002] High-sensitivity and high-specificity DNA detection technology has been widely used in disease diagnosis, drug development, food safety, environmental monitoring, etc. In recent years, DNA detection technology tends to be miniaturized and simplified. The improvement of DNA detection technology, the reduction of cost and easier process control are the current development trends. Compared with early DNA detection methods such as colorimetry, gel electrophoresis, and ultraviolet spectroscopy, DNA sensors using fluorescence methods have attracted more and more attention due to their high sensitivity, low cost, and portability. [0003] Document 1 (Li Hongye, Fan Shuguo, Liu Yule, Chen Gang, Wang Huanyu, DNA detection by improved polyacrylamide gel electrophores...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N21/33G01N21/75
Inventor 孔金明胡伟文何玟辉顾淑梅梅亚琦
Owner NANJING UNIV OF SCI & TECH
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